Examination of Mechanism of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Reveals Unexpected Role for Dynamic Tyrosine

Huang, Xinyi; Hernick, Marcy

doi:10.1074/jbc.m111.320184

Cited by 15 publications

(42 citation statements)

References 40 publications

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“…12 We previously confirmed the importance of the Tyr142 and His144 side chains in GlcNAc recognition and turnover using site-directed mutagenesis and enzyme kinetic experiments. 16 Importantly, we have now also confirmed the importance of the Arg68 and Asp95 side chains for GlcNAc recognition and turnover using similar site-directed mutagenesis and enzyme kinetic experiments ( Figure 4 and Table 1). Removal of either side chain with the D95A and R68A mutants results in a complete loss of MshB activity with the GlcNAc substrate, confirming their importance for molecular recognition and turnover by MshB.…”

Section: ■ Discussionsupporting

confidence: 80%

Molecular Determinants of N-Acetylglucosamine Recognition and Turnover by N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB)

Huang

Hernick

2015

Biochemistry

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Actinobacteria such as Mycobacterium tuberculosis use the unique thiol mycothiol (MSH) as their primary reducing agent and in the detoxification of xenobiotics. N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) is the metal-dependent deacetylase that catalyzes the deacetylation of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside, the committed step in MSH biosynthesis. We previously used docking studies to identify specific side chains that may contribute as molecular determinants of MshB substrate specificity [Huang, X., and Hernick, M. (2014) Biopolymers 101, 406-417]. Herein, we probe the molecular basis of N-acetylglucosamine (GlcNAc) recognition and turnover by MshB using a combination of site-directed mutagenesis and kinetic studies (mutants examined, L19A, E47A, R68A, D95A, M98A, D146N, and F216A). Results from these studies indicate that MshB is unable to catalyze the turnover of GlcNAc upon loss of the Arg68 or Asp95 side chains, consistent with the proposal that these side chains make critical hydrogen bonding interactions with substrate. The activity of the D146N mutant is ∼10-fold higher than that of the D146A mutant, suggesting that the ability to accept a hydrogen bond at this position contributes to GlcNAc substrate specificity. Because there does not appear to be a direct contact between Asp146 and substrate, this effect is likely mediated via positioning of other catalytically important residues. Finally, we probed side chains located on mobile loops and in a hydrophobic cavity and identified two additional side chains (Met98 and Glu47) that contribute to GlcNAc recognition and turnover by MshB. Together, results from these studies confirm some of the molecular determinants of GlcNAc substrate specificity by MshB, which should aid the development of MshB inhibitors.

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Section: ■ Discussionsupporting

confidence: 80%

Molecular Determinants of N-Acetylglucosamine Recognition and Turnover by N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB)

Huang

Hernick

2015

Biochemistry

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“…The GalB pK a values for both the enzyme-substrate complex and the free enzyme/substrate are unaffected by the metal cofactor utilized. Bell-shaped profiles for pH dependence on k cat /K m for Zn 2ϩ cofactor reactions are observed with the PIG-L deacetylases MshB (pK a values 7.3 and 10.5) and BshB (pK a values 6.5 and 8.5), which are similar to GalB (45,63). In both MshB and BshB, a minimal or no effect is observed on either pK a when utilizing Co 2ϩ as the cofactor, and in MshB neither Fe 2ϩ , Ni 2ϩ , nor Mn 2ϩ affected the pK a values (45,63).…”

Section: Discussionmentioning

confidence: 60%

“…Bell-shaped profiles for pH dependence on k cat /K m for Zn 2ϩ cofactor reactions are observed with the PIG-L deacetylases MshB (pK a values 7.3 and 10.5) and BshB (pK a values 6.5 and 8.5), which are similar to GalB (45,63). In both MshB and BshB, a minimal or no effect is observed on either pK a when utilizing Co 2ϩ as the cofactor, and in MshB neither Fe 2ϩ , Ni 2ϩ , nor Mn 2ϩ affected the pK a values (45,63). In the proposed GalB and PIG-L deacetylase reactions, the metal contributes to the mechanism by positioning and likely lowering the pK a of the catalytic water.…”

Section: Discussionmentioning

confidence: 60%

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Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

Mazurkewich

Brott

Kimber

et al. 2016

Journal of Biological Chemistry

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The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of lignobiomass. The gallate degradation pathway of Pseudomonas putida KT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identified CHM hydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-acetylglucosamine deacetylase family; GalB is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereospecificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn 2؉ cofactor; however the enzyme is capable of using Fe 2؉ and Co 2؉ with similar efficiency. The general base aspartate in the PIG-L deacetylases is an alanine in GalB; replacement of the alanine with aspartate decreased the GalB catalytic efficiency for CHM by 9.5 ؋ 10 4 -fold, and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependence studies of the wild type and variant enzymes suggested roles for Glu-48 and His-164 in the catalytic mechanism. A comparison with the PIG-L deacetylases led to a proposed mechanism for GalB wherein Glu-48 positions and activates the metal-ligated water for the hydration reaction and His-164 acts as a catalytic acid.

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“…In addition, in the X-ray structure there are two water molecules that coordinate to the Zn ion, one of which establishes a hydrogen bond with Asp15, the proposed general base catalyst (Newton et al, 2000). Tyr142, His144, and Asp146 have also been suggested to play an important role in the catalytic mechanism, although details are not clear (Huang & Hernick, 2012).…”

Section: Mshb (Rv1170)mentioning

confidence: 98%

Efficient Calculation of Enzyme Reaction Free Energy Profiles Using a Hybrid Differential Relaxation Algorithm

Romero¹,

Martin²,

Ramírez³

et al. 2015

Combined Quantum Mechanical and Molecular Mechanical Modelling of Biomolecular Interactions

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Examination of Mechanism of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Reveals Unexpected Role for Dynamic Tyrosine

Cited by 15 publications

References 40 publications

Molecular Determinants of N-Acetylglucosamine Recognition and Turnover by N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB)

Molecular Determinants of N-Acetylglucosamine Recognition and Turnover by N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB)

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

Efficient Calculation of Enzyme Reaction Free Energy Profiles Using a Hybrid Differential Relaxation Algorithm

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